1. Field of the Invention
This invention relates to preparative-scale separation of components by electrophoresis. More particularly, method and apparatus for altering the initial composition of a feed solution by electrophoretic separations using a resolving agent to dynamically alter the sign of the effective mobility of the band of one or more selected sample components in the feed solution is provided.
2. Description of Related Art
The ability to conduct preparative-scale chromatographic enantiomer separations has improved significantly during the last decade. Once the fundamentals of nonlinear chromatography became well understood and adequate hardware became readily available, many batch-wise nonlinear chromatographic separations were converted to continuous separations, using a simulated moving bed approach, to achieve higher productivities. [Juza, M. et al., Trends in Biotech. 2000, 18, 108–118] Mirroring these developments, as understanding of capillary electrophoretic enantiomer separations improved, preparative-scale electrophoretic enantiomer separations were attempted. The appearance of a new, continuous, free-flow electrophoretic units, such as the “Octopus,” permitted exploration of the conversion of batch-type electrophoretic separations to continuous, preparative-scale separations that can alter the initial composition of the feed solution. These developments are more fully explained in a recent paper co-authored by the inventor [“Use of Single-Isomer, Multiply-Charged Chiral Resolving Agents for the Continuous, Preparative-Scale Electrophoretic Separation of Enantiomers Based on the Principle of Equal-But-Opposite Analyte Mobilities,” Electrophoresis 2000, 21, 1019–1026], which is hereby incorporated by reference herein.
The Octopus unit is illustrated in FIG. 1 (Prior Art). There is a continuous, laminar flow of the separation medium, orthogonal to the electric field, through shallow, rectangular electrophoresis chamber 10 of the unit. The sample is continuously fed at inlet 12, above inlet ports 14 where the separation medium enters the unit, either at the center or at one of the sides of the chamber. The separated components, dissolved in the separation medium, are collected through sampling ports 16 as they leave the separation chamber. The sampling ports (normally including 96 ports) provide a lateral spatial resolution of about 1 mm per collection port across the 100 mm wide separation chamber. Recent studies indicated that the reproducibility and long-term stability of the separation patterns obtained in the Octopus unit were satisfactory.
The Octopus unit is well suited for preparative-scale, continuous isoelectric focusing separations because the well known isoelectric focusing mechanism successfully counters most of the flow-related band broadening mechanisms. The Octopus unit has been successfully used for the preparative-scale isoelectric focusing separation of the enantiomers of dansyl phenylalanine with 30 mM hydroxypropyl β-cyclodextrin as chiral resolving agent in binary Bier buffers. [P. Glukhovskiy et al., Anal. Chem., 1999, 71, 3814–3820] The Octopus unit has also been used for the much more difficult, continuous free-flow and intermittent-flow electrophoretic separation of the enantiomers of methadone with non-charged hydroxypropyl β-cyclodextrin as the chiral resolving agent. [P. Hoffmann et al., Anal. Chem., 1999, 71, 1840–1850] In both of these flow modes, the cationic methadone enantiomers were injected at the anodic side of the separation chamber (opposite to fraction collection ports 19–20), and were collected, partially separated, in fractions 52–96 (continuous flow mode) and fractions 72–96 (intermittent flow mode). This means that in the continuous flow mode the available separation distance was only twice as large (about 80 ports wide) as the band width of each enantiomer (about 44 ports wide) resulting in an alteration of the initial composition of the feed solution. The situation was a little better in the intermittent flow mode, where the available separation distance (about 80 ports wide) was four times as large as the band width of each enantiomer (about 20 ports wide) resulting, once again, in an alteration of the initial composition of the feed solution. Clearly, one would need a much larger separation distance (much wider separation chamber) if one wanted to completely eliminate the overlap of the enantiomer bands to not only alter the initial composition of the feed solution, but recover each enantiomer in pure form.
In general, the separation of two like-charged analyte ions of similar, but not identical effective mobilities (derived either from strong electrolytes or weak electrolytes) by electrophoresis typically requires the use of long migration distances and/or large applied electric potentials. While these requirements can be fulfilled relatively easily in capillary electrophoresis for analytical-scale separations, they are often difficult or impossible to meet in preparative-scale separation.
Hydrodynamic flows or electroosmotic flows have been utilized to shift the observed mobilities of at least one of the analytes to be separated, as described, e.g., in the paper by B. A. William and Gy. Vigh, “The Use of Hydrodynamic Counterflow to Improve The Resolution of the Minor Component in the Capillary Electrophoretic Analysis of Enantiomers.” Enantiomer 1 (1996) 183. However, due to the frequently poor temporal stability of the electroosmotic flow, and the extra band broadening created by the laminar hydrodynamic flow, these approaches are not conducive to efficient preparative-scale separations. What is needed is a method for use in preparative-scale electrophoresis that can alter the initial composition of the feed solution over relatively short migration distances.